Characterization and Comparison of Mesenchymal Stem Cell-Derived Exosome Isolation Methods using Culture Supernatant.

Exosomes are extracellular endosomal nanoparticles, which are formed under complex processes during the formation of multivesicular bodies. They are also achieved from conditioned media of a variety of cell types, especially mesenchymal stem cells (MSCs). Exosomes can modulate intracellular physiological actions via signaling molecules on the surface or secretion of components to the extracellular spaces. Furthermore, they are potentially used as crucial agents for cell-free therapy; however, their isolation and characterization can be challenging. In the current study, two methods of exosome isolation have been characterized and compared using a culture media of adipose-derived mesenchymal stem cells, namely ultracentrifugation and a commercial kit; moreover, the efficiency of these two methods was highlighted in this study. Two different isolation methods of exosomes from MSCs were used to compare the efficiency of exosomes. For both isolation methods, transmission electron microscopy, dynamic light scattering (DLS), and bicinchoninic acid (BCA) assay have been performed. The electron microscopy and DLS indicated the presence of exosomes. Moreover, the kit and ultracentrifugation isolates contained approximately comparable amounts of protein measured by the BCA. Overall, the two isolation methods had similar performances. Although ultracentrifugation is used as a gold standard for exosome isolation, the commercial kit has some advantages and can be applied alternatively according to its cost-effectiveness and time-saving properties.

Use of cellular exosomes as a new carrier in breast cancer gene therapy.

BACKGROUND: Breast cancer is recognized as a major clinical challenge in gynecological diseases worldwide. Exosomes are small vesicles derived from multicellular bodies that are secreted by many cells into the extracellular environment and thus participate in intercellular communication through the transfer of genetic information such as encoded and non-encoded RNAs to target cells. Tumor-derived exosomes are thought to be a rich source of microRNAs (miRs) that can regulate the function of other cancer cells in the tumor microenvironment. However, the exact mechanisms by which tumor cell-derived exosomes affect their neighboring cells, as well as the bio-logical function of exosomal miRs in receptor cells, are not well understood. MATERIALS AND METHODS: In this study, after the overexpression of MiR-205 in breast cancer cells (MDA-MB-231 class), cell-derived exosomes were successfully isolated and characterized by electron microscopy and dynamic light scattering. RESULTS: The determination of MiR-205 expression levels in exosomes secreted from engineered cells confirmed the high expression of this miR in exosomes. It was also found that the treatment of tumor exosomes carrying this miR had an apoptotic induction effect and also had a significant effect on reducing the expression of Bcl-2 gene transcript in a time-dependent manner in breast cancer cells (P < 0.001). CONCLUSION: Overall, this study suggests that exosomal transfer of tumor suppressor miRs to cancer cells could be a suitable platform for nucleic acid transfer to these cells and be highly effective in cancer treatment.

Mesenchymal stem cell-derived exosomes: A novel potential therapeutic avenue for cardiac regeneration.

Coronary artery diseases (CADs) represent a significant cause of death worldwide. During recent decades the rate of cardiovascular mortality has been declined as a result of modern medicine and surgery. However, despite the fact that cardiac cells, including cardiomyocytes (CMCs), vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC), can be regenerated by cardiac adult stem cell, the regenerative capacity of these cells are limited and inadequate to functionally regenerate heart damaged tissue. Thus, growth reserve of the heart fails to restore the structural integrity of the myocardium after infarction and healing is associated with scar formation. An explanation for this is that cardiac reside stem cells are present throughout the infarction site but die rapidly by apoptosis. Furthermore, microenvironment surrounding the damage site is not promising for the cells survival and renewal. Hence, recent advances in the stem cell therapy have emerged as an attractive approach to replace the lost cells. In this context, mesenchymal stem cells (MSCs) has considered as one of the most promising candidates for regeneration of cardiac cells, lost upon injury. The regenerative capacity of MSCs has primarily been centered on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac cells. However, experimental and clinical observations so far have failed to establish if this differentiated is considerably relevant to MSCs cardiac regenerative properties. Recent reports have suggested that these therapeutic properties, at least in part, are mediated by paracrine factors released from MSCs. This review provides a concise summary of current evidences supporting the paracrine hypothesis of MSCs. In particular, the scope of this review focuses on the role of MSC-derived exosome (MSC-EXs) as a therapeutic modality for the treatment of CADs, particularly ischemic myocardial dysfunctions.

Therapeutic application of mesenchymal stem cell-derived exosomes: A promising cell-free therapeutic strategy in regenerative medicine.

Mesenchymal stem cells have emerged as promising therapeutic candidates in regenerative medicine. The mechanisms underlying mesenchymal stem cells regenerative properties were initially attributed to their engraftment in injured tissues and their subsequent transdifferentiation to repair and replace damaged cells. However, studies in animal models and patients indicated that the low number of transplanted mesenchymal stem cells localize to the target tissue and transdifferentiate to appropriate cell lineage. Instead the regenerative potential of mesenchymal stem cells has been found - at least in part - to be mediated via their paracrine actions. Recently, a secreted group of vesicles, called "exosome" has been identified as major mediator of mesenchymal stem cells therapeutic efficacy. In this review, we will summarize the current literature on administration of exosomes released by mesenchymal stem cells in regenerative medicine and suggest how they could help to improve tissue regeneration following injury.

Introducing Dendrosomal Nanocurcumin as a Compound Capable of in vitro Eliminating Undifferentiated Stem Cells in Cell Therapy Practices.

One of the major obstacles needed to be overcome before using cell therapy for clinical purposes is the high probability of tumor formation in patients who receive the transplants, as undifferentiated stem cells (SCs) have the potential to form teratomas/teratocarcinoma in xenotransplants. In this study the ability of dendrosomal nanocurcumin (solublized curcumin using a biodegradable non-toxic nano-carrier) to affect undifferentiated/hazardous cell, and hence increasing the safety of cell therapy (particularly in diabetes type I) by mesenchymal stem cells (MSCs) was examined. The results showed that after completion of differentiation of human mesenchymal stem cells (hMSCs) into insulin producing cells (IPCs), the expression level of insulin increases, although there remains a minority of undifferentiated cells which still express nestin (gene which is expressed in progenitor stem cells of IPCs). It indicates the emergence of a heterogeneous population containing undifferentiated and differentiated cells. Furthermore our data demonstrated that the expression level of p53 decreases during differentiation of hMSCs into IPCs which implies a more favorable microenvironment for tumor formation following the transplantation of such a heterogeneous population. After treatment with dendrosomal nanocurcumin, nestin expression eliminated, however no significant effect on the expression and secretion of insulin was observed. Together our data shows that dendrosomal nanocurcumin have the ability to affect residual undifferentiated stem cells after the completion of differentiation of MSCs induced to differentiate into IPCs; while it exerts no significant harmful effect on the survival and function of differentiated cells. With regard to the obtained results in this study, exploiting dendrosomal nanocurcumin, after completion of induced differentiation of stem cells and prior to the transplantation step, can be suggested as a very efficient, safe and cost-effective method to eliminate the residual undifferentiated stem cells in cell therapy practices in order to considerably decrease the risk of tumor development following transplantation.